During development, cranial Neural Crest Cells (cNCCs) play major roles in establishing craniofacial morphology and determining its species-specific variation. To understand human distinctive features it is imperative to study human cNCCs and their derivatives in addition to cNCCs from model organisms. Since human NC formation occurs at 3 to 6 weeks of gestation and is largely inaccessible to genetic studies, we have established a human pluripotent stem cell-based cNCC differentiation model in the dish with high relevance to craniofacial development. Moreover, we have extended our model to chimpanzee cNCCs, allowing us to identify molecular features that distinguish human cNCCs from those of our closest evolutionary cousins. First, we propose to characterize epigenetic landscapes and transcriptomes of human and chimpanzee cNCCs and to identify conserved and species-specific cis-regulatory elements utilized by this unique cell type. Since chromatin modification maps from NCCs of any organism are not yet publicly available beyond our report, we will generate chromatin marking profiles from a cohort of human and chimp post- migratory cNCCs, complemented with transcriptome analyses. Thus, we will create reference epigenomes that will be annotated for active and poised enhancers and promoters. We have already identified over 2000 regulatory elements that show strong species-specific bias in their chromatin marks in human versus chimpanzee, arguing that human-specific cNCC molecular features do exist and may underlie human-specific craniofacial divergence. These datasets will provide a rich resource for future investigations of the transcriptional and epigenetic basis of human craniofacial evolution, development, and disease. Second, we will analyze candidate human-specific craniofacial enhancer activity in vivo. To this end, transgenic reporter assays in mouse embryos will be used to analyze the activity of 50 human regulatory elements that either gained or lost active enhancer signature in human cNCCs, as compared to the 50 orthologous chimpanzee regions. Thus, we will generate a validated set of human-specific craniofacial enhancers that can be further explored in mechanistic studies. For 10 selected human-specific enhancers exhibiting gain or loss of activity, interesting activity patterns, or relevant associatin with craniofacial development or disease in humans, we will utilize BAC recombineering to further develop founder transgenic lines that will be distributed to the craniofacial community.